Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. I. Adrenocorticotropin (ACTH) and alpha-melanotropins (alpha-MSHs)

We have studied the post-translational processing of POMC-derived peptides during fetal monkey development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Pituitary tissues obtained from fetal monkeys ranging from Gestational Day 50 to 155 were fractionated and analyzed for ACTH- and alpha-MSH-related peptides and compared to adult forms. Extracts of whole pituitary from Fetal Days 50 and 55 contained ACTH(1-39) and very small amounts of CLIP (corticotropin-like intermediate-lobe peptide; ACTH(18-39))-like immunoactivity. Acetylated alpha-MSHs were not detectable at Day 50. alpha-MSHs were barely detectable at Day 55. By Day 65, when pituitary lobes were separable, small amounts of des-, mono-, and diacetyl alpha-MSH were detectable in NIL extracts, but not in anterior lobe extracts. ACTH(1-39) levels were negligible when compared to increasing alpha-MSHs through Fetal Day 80 to 155 in the intermediate lobe. The CLIP immunoactivity was negligible in Day 80 and adult anterior lobe extracts. Thus, lobe-specific proteolytic processing of ACTH-related peptides was well established by midterm gestation. Marked increases of alpha-N- and alpha-N,O-acetylated forms of alpha-MSHs were detected during middle and late stage fetal development. Diacetyl alpha-MSH was the predominant form of alpha-MSH in adult NIL extracts. No acetylated alpha-MSHs were found in anterior lobe tissues, thus adult anterior lobe extracts contained almost exclusively ACTH(1-39). However adult NIL extracts contained two distinct forms of CLIP-related immunoactivity. Therefore changes in post-translational processing patterns of ACTH-related and alpha-MSH-related peptides continued to some extent, postnatally. These data indicate that marked changes in post-translational processing of POMC-derived ACTH-related products occur during the first half of monkey gestation.     

In vitro biosynthesis of gamma-lipotropic hormone.

Sheep gamma-lipotropic hormone (gamma-LPH) is a pituitary polypeptide made of 58 amino acids and is formed of the first 58 residues of beta-lipotropic hormone (beta-LPH). The C-terminal portion (41-58) of gamma-LPH is identical with the structure of beta-melanophore-stimulating hormone (beta-MSH). We hypothetized in 1967 that beta-LPH could be the biological precursor of beta-MSH and that gamma-LPH could be an intermediate compound. We demonstrated in 1974 that beta-LPH is actively synthesized in the bovine pituitaries. We now studied the biosynthesis of gamma-LPH by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated gamma-LPH from the other radioactive proteins with a method previously described. We characterized the radioactive proteins by ion-exchange chromatography, gel filtration and polyacrylamide gel electrophoresis. Our results show that radioactive gamma-LPH was actively synthesized. This gamma-LPH has all the chemical characteristics of nonradioactive gamma-LPH. However, in the conditions used, we were unable to demonstrate biosynthesis of beta-MSH. These results suggest that gamma-LPH is biosynthesized more slowly than beta-LPH and that the conversion into beta-MSH, if it exists, is a slow or subactive process in the species studied.     

Autonomous beta-endorphin secretion from the pituitary neurointermediate lobe: in vivo studies

The existence of independent control mechanisms of beta-endorphin (beta-EP) secretion from the anterior (AP) and intermediate (NIL) pituitary lobes is now ascertained. The aim of this study was to evaluate the effect of surgical separation from the hypothalamus of the two pituitary lobes on beta-EP secretion. Two experimental models of surgical hypothalamo-pituitary disconnection were used: 1) rats with ablation of the medial basal hypothalamus (MBH); 2) rats bearing two entire ectopic pituitaries or two anterior pituitaries (APs) only, transplanted under the kidney capsule. In rats with MBH-ablation plasma beta-EP levels were significantly higher than in sham-operated controls. Plasma beta-EP levels increased in rats transplanted with entire pituitaries 3 days after surgery and were still elevated after 1 week. In rats transplanted with APs only, no significant beta-EP changes in plasma were evident. In both experimental conditions no significant difference was present in beta-LPH plasma levels. Concentrations of beta-EP in the ectopic NILs decreased gradually after transplantation. In all these results indicate that that NIL but not the AP is capable, when is disconnected from the hypothalamus, or secreting autonomously beta-EP.     

An investigation of N-terminal pro-opiocortin peptides in the rat pituitary

Extracts of neurointermediate lobe (NIL) and anterior lobe (AL) of the rat pituitary, and material released from perfused rat pars distalis (PD) and pars intermedia (PI) cells were gel chromatographed and monitored using three antisera, each recognizing different regions of the non-corticotropin (ACTH)-lipotropin (LPH) portion of pro-opiocortin (POC). Two peaks (termed N-POC I) which emerged close to the elution position of rat beta-LPH were detected. The first peak was reduced significantly in the PI. Two smaller N-POC fragments which eluted near beta-endorphin were detected only in extracts and secretions of intermediate lobe tissue. One peak cross-reacted in the gamma 3-melanotropin (MSH) assay (N-POC III) whereas the other peak possessed amino (N)-terminal N-POC immunoreactivity (N-POC II). The results demonstrated differences in the distribution and nature of N-POC peptides released and extracted from the PD and PI of the rat pituitary, and suggest that the enzymic processing of N-POC is different in the two pituitary lobes.     

Increase of beta-endorphin concentrations in the plasma and pituitary neuro-intermediate lobe of the rat on the afternoon of proestrus

Beta-endorphin (beta-EP) concentrations in the plasma and the anterior and neuro-intermediate lobes of the pituitary (AP and NIL) were quantitated by radioimmunoassay (RIA) and gel filtration chromatography in female rats at 1000, 1400, and 1900 h on the day of proestrus and diestrus day-1. There were no significant changes in beta-EP in the plasma, AP, or NIL on diestrus day-1. On proestrus, beta-EP in the plasma and NIL, but not the AP, increased significantly from 1000 to 1400 h and returned to basal levels by 1900 h. The time course of this increase of beta-EP in the NIL and plasma is consistent with the temporal sequence of the prolactin and gonadotropin surges on the afternoon of proestrus, suggesting that beta-EP in the NIL may be involved in the regulation of these neuroendocrine events.     

Chronic ethanol treatment alters the biosynthesis of beta-endorphin by the rat neurointermediate lobe

Tolerance to ethanol was induced in male Sprague-Dawley rats (225-250 g) by chronic feeding with a liquid diet containing 6.5% ethanol (v/v). Control rats were pair-fed with a liquid diet in which the ethanol was replaced by an equicaloric concentration of sucrose. Immediately following sacrifice of the animals the neurointermediate lobes (NIL) were removed and incubated with [3H]phenylalanine. The biosynthesized proopiomelanocortin (POMC), beta-lipotropin (beta-LPH), and beta-endorphin (beta-EP) were purified by immunoprecipitation with an antiserum to beta-EP and analyzed by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Alcohol treatment for 3 days had no effect on the degree of incorporation of [3H]phenylalanine into POMC, beta-LPH, and beta-EP but treatment for either 15 or 21 days increased the incorporation of [3H]phenylalanine into all three peptides. Ethanol treatment also increased the beta-endorphinlike immunoreactivity (beta-EPLIR) found in the incubation medium, but no significant change was observed in the beta-EPLIR extracted from the NIL either immediately after sacrifice or after 3 h of incubation of the NIL. However, a significant decrease of beta-EPLIR was found in the anterior lobes of rats treated with ethanol for 21 days. Furthermore, the beta-EPLIR in the serum of alcohol-treated rats was significantly higher than in the serum of their corresponding controls. These results indicate an effect of ethanol on the endorphin system and are consistent with the suggestion that endorphins may be mediators of some of the ethanol effects.     

Biosynthesis and processing of pro-opiomelanocortin-derived peptides during fetal pituitary development

We have investigated the molecular weight forms of pro-opiomelanocortin (POMC)-derived peptides present in rat pituitaries during fetal and early postnatal development (embryonic Day 14 to 3 day neonate). At all early ages examined, the major immunoreactive form of corticotropin (ACTH) was POMC. Only during late fetal and early postnatal stages did progressively larger amounts of 4.5K ACTH, a major POMC processing end product, appear. This form was found almost exclusively in isolated anterior lobes. In contrast, 3.5K size endorphin(s), another POMC derivative, were present in whole glands even at early stages (Day 14), and were the major POMC derivative(s) found in isolated intermediate-posterior lobes of older fetuses. Despite the early appearance of 3.5K endorphin(s), α-MSH did not appear until Day 19 and was detected only in isolated intermediate-posterior lobes. We have also cultured dispersed fetal pituitary cells in the presence of radioactive amino acids. After immunoprecipitation using affinity-purified antisera, followed by fractionation of the radiolabeled products, we found that POMC biosynthesis does occur in cultures of Day 14 embryonic pituitary cells, and that the major POMC-derived end product produced is 3.5K size endorphin(s). These findings demonstrate that POMC is synthesized at least by Day 14 of rat pituitary development and that lobe-specific processing characteristic of the corresponding adult lobe is apparent at the earliest stages that the lobes can be separated. The presence of 3.5K-sized endorphins at early ages is consistent with the possibility that POMC synthesis first occurs in the intermediate lobe. The noncoordinate appearance of α-MSH, 1–39 ACTH, and endorphins implies that the activities of certain cleavage enzymes and acetylation enzymes responsible for lobe-specific post-translational POMC processing may be expressed at different times during development.     

An immunocytological study of the adult crab-eating macaque (Macaca fascicularis) pituitary and its cytological differentiation during fetal life

The pituitaries of adult and fetal crab-eating macaques (Macaca fascicularis) have been studied by immunofluorescence using 15 antibodies against most of the known hormones in the adenohypophysis. The antibodies used were first checked on adult pituitaries for their cross-specificity with macaque pituitary hormones. We found five types of endocrine cells reacting positively, according to the biochemical relation of the molecules evidenced with one or more of the antibodies used. The sequential appearance of the various hormones in the cells of the anterior and intermediate lobes was then determined. The first hormones evidenced at day 45 of pregnancy were ACTH, beta-MSH, beta- and gamma-LPH and alpha- and beta-endorphins. alpha-MSH appeared at day 48 and STH at day 51. The glycoprotein hormones, LH, FSH and TSH, appeared at day 57 but the thyrotropes and gonadotropes did not attain their adult characteristics (staining intensity, morphology, density and distribution in the pituitary) until days 71 and 93, respectively. Prolactin was only found beginning at day 93 of pregnancy. The different specificity tests applied to the pituitary of the macaque, as well as to that of other vertebrates, show that the antibodies used have good specificity. A comparison of the dates at which the fetal pituitary gonadotropes appear in the macaque and the results of a developmental study of the external genital organs in that species indicated that the pituitary gonadotropic function is only established after somatic sex differentiation, which would thus probably occur independently.     

Content of beta-LPH and its fragments (including endorphins) in anterior and intermediate lobes of the bovine pituitary gland

Radioimmunoassays (RIAs) specific for β-LPH_1–47, β-endorphin, α-MSH and β-MSH have been used to identify immunoreactive components in acid extracts from anterior and intermediate lobes of bovine pituitary gland after separation by chromatography on Sephadex G-50. When components in extracts of both lobes, eluting at the same position, were measured with the β-endorphin and β-LPH_1–47 RIA systems, marked quantitative differences were seen. The main components reacting with the β-LPH_1–47 system in anterior pituitary extract co-migrated with β-LPH and γ-LPH while in the intermediate lobe, the main immunoreactive component eluted at a position slightly later than β-endorphin. When the β-endorphin RIA system was used, relatively low amounts of immunoreactive material co-migrating with β-endorphin were seen in the anterior lobe extract while a highly predominant peak eluting at a position slightly later than β-endorphin was observed in intermediate lobe extract. Some β-MSH was seen in the intermediate lobe. These date indicate that the processing of β-LPH is markedly different in the anterior and intermediate bovine pituitary lobes: β-endorphin immunoreactive material predominates in the intermediate lobe whereas β-LPH and γ-LPH predominate in the anterior lobe.     

Beta-endorphin concentrations in the hypothalamus, pituitary and plasma of streptozotocin-diabetic rats with and without insulin substitution therapy

The concentrations of beta-endorphin like immunoreactivity (beta-END) in the hypothalamus, pituitary and plasma were studied in rats of either sex, one month after induction of diabetes by single iv injection of streptozotocin. As controls, both normal and undernourished rats, weight-matched with diabetic rats, were used. Diabetic male and female rats had a marked depletion of beta-END stores in the hypothalamus and neurointermediate lobe (NIL) but not in the anterior pituitary. Depletion of beta-END was reversed to normal by insulin replacement therapy. Severe undernourishment was not as effective as diabetes to reduce beta-END stores in the hypothalamus and NIL. A significant reduction of beta-END was observed only in the NIL of undernourished female rats. Plasma beta-END and beta-lipotropin (beta-LPH) concentrations were not significantly altered in diabetic rats. These results indicate that the lack of insulin may affect beta-END synthesis in the hypothalamus and NIL.     

Chemistry and biosynthesis of pro-opiomelanocortin

Studies of lipotropins, melanotropins and endorphins on one hand, and of adrenocorticotropin on the other, has given rise to the concept of a multipotent precursor molecule recently renamed proopiomelanocortin. The preferential sites of cleavage of the precursor to produce its biologically active components are made of pairs of basic amino acid residues as described for the biosynthesis of beta-MSH and pro-insulin. Such structural feature is also found in other pro-hormone molecules. Pulse chase experiments and secretory studies carried out in both anterior and intermediate lobes of rat pituitary glands revealed the transformation of different forms of the precursor into different end-products, the anterior lobe producing preferentially ACTH and beta-LPH while the intermediate produces mainly the alpha-MSH and beta-endorphin. The multiple forms of precursors seem to differ in their carbohydrate content although at least two different gene products are still possible. The presence of similar peptides in the hypothalamus makes it highly probable that neuropeptides are biosynthesized with similar process. Thus the model of beta-LPH precursor, proposed as early as in 1967, is now applicable to the biosynthesis of all other neuropeptides. Major advances in this field are expected in the 1980s.     

α-N-Acetyl β-Endorphins in the Pituitary: Immunohistochemical Localization Using Antibodies Raised Against Dynorphin(1-13)

Abstract: Intense immunohistochemical staining of the intermediate lobe of the pituitary was observed by using an antiserum raised against synthetic dynorphin(1-13) treated with a water-soluble carbodiimide (CDI). Subsequent studies showed that the immunostaining was blocked by preincubation of the antiserum with acetylated derivatives of both β-endorphin and dynorphin(1-13) as well as by CDI-treated dynorphin(1-13), but only weakly by authentic dynorphin(1-13). Neither nonacetylated β-endorphin nor any other fragments of the ACTH/endorphin precursor blocked the immunostaining of the intermediate lobe. Analysis of the CDI-treated dynorphin(1-13) used as an antigen showed that most of the peptide was acetylated at primary amino groups. CDI treatment of dynorphin(1-13) results in the formation of an acetyl derivative because the commercially available peptide is supplied as the acetate salt. The antibodies responsible for the intermediate lobe staining were isolated by affinity chromatography, using a column containing partially purified intermediate lobe extract linked to an affinity resin and a radioimmunoassay (RIA) was developed with CDI-treated dynorphin(1-13) used as a trace and as a standard. Competition studies showed 0.5-1% cross-reactivity with α-N-acetyl β-endorphin(1-31), α-N-acetyl β-endorphin(1-27), and totally acetylated β-endorphin(1-31). Nonacetylated β-endorphins did not cross-react. Posterior-intermediate lobe extracts from rat and beef were fractionated by gel filtration. Rat posterior-intermediate lobe extracts were also fractionated by cation-exchange chromatography. Fractionated extracts were analyzed by RIAs for β-endorphin, CDI-treated dynorphin(1-13), and authentic dynorphin(1-13). The results suggested that the peptides responsible for the intermediate lobe staining were mainly four different derivatives of β-endorphin bearing an acetyl group at the amino terminus. No immunostaining was seen in the posterior and anterior lobes of the pituitary. This suggests that the intermediate lobe is the main source of acetylated β-endorphins in the pituitary.     

The preferential release of beta-endorphin from the anterior pituitary lobe by corticotropin releasing factor (CRF)

Although a number of investigators have shown that release of ACTH is accompanied by the release of Beta-endorphin (beta-End) and Beta-lipotropin (beta-LPH), the proportion of the latter two peptides released with stress or by CRF is unclear. To evaluate directly the release of beta-End versus beta-LPH from the anterior lobe, we used molecular sieving of plasma and subsequent radioimmunoassay to measure release of both beta-End and beta-LPH into plasma after thirty minutes of inescapable intermittent footshock. We found a substantial increase in circulating beta-End which appears to be of anterior lobe origin. The beta-End does not appear to represent peripheral conversion of beta-LPH to beta-End since the ratio of beta-LPH:beta-End released remained constant between five and thirty minutes of stress, and the rate of disappearance of beta-LPH is slower than the rate of disappearance of beta-End following the termination of stress. Further confirmation of these findings was obtained by examining the POMC derived peptides released by pituitary cell suspensions in the presence and absence of oCRF. While unstimulated release consisted of equal proportions of beta-End and beta-LPH, stimulation of the anterior lobe cell suspensions with oCRF resulted in the release of two-fold more beta-End than beta-LPH.     

Identification of carp proopiomelanocortin-related peptides and their effects on phagocytes

We report the immunomodulating effects of proopiomelanocortin (POMC)-related peptides on phagocytic cells in carp. The complete amino acid sequences of two carp POMCs (I and II) were deduced from the nucleotide sequences after cDNA cloning. Both POMCs consist of 194 amino acids (91% sequence identity) including identical alpha-melanotropin (MSH) and beta-endorphin (EP). All hormonal peptides derived from two POMCs were identified by mass spectrometry after separation by high-performance liquid chromatography of an acid-acetone extract from a single pituitary. These peptides were alpha-MSH, N-Des-Ac-alpha-MSH, di-Ac-alpha-MSH, beta-MSH I, beta-MSH-II, N-Ac-beta-EP(1-29), corticotropin-like intermediate lobe peptide I and II and N-terminal peptide of POMC I and II. The immunomodulating effects of synthetic MSHs and EPs on phagocytic cells from carp head kidney were examined. Di-Ac-alpha-MSH, beta-MSH I, N-Ac-beta-EP(1-29) and beta-EP(1-29) increased the production of superoxide anion at 0.1-100 ng ml-1 for these MSHs and 1-100 ng ml-1 for EPs in RPMI 1640 medium.     

Cytoimmunological and histochemical study of cat adenohypophyseal cells, made fluorescent by the Falck and Hillarp technic]

In the Cat, after Falck and Hillarp method, all the fluorescent cells of the PI and the anterior lobe of adenohypophysis can be revealed with specific anti-sera to ACTH(1-24), ACTH(17-39), bovine beta-MSH and porcine beta-LPH. With the lead hematoxyline staining method, two types of cells are recognizable in the anterior lobe, in which the non hormonal constituents of the granules must be different.     

Glucocorticoid inhibition of immunoreactive beta-endorphin release from the anterior lobe of the rat pituitary: in vitro and in vivo studies

Glucocorticoid control of pituitary beta-endorphin (beta-END) release was investigated in vitro and in vivo. Cultured cells of both rat anterior (AL) and neurointermediate (NIL) lobe released beta-END-like immunoreactivity (beta-END-LI) in response to epinephrine (10(-7) M); however, only the response of AL cells was prevented by corticosterone (10(-8)-10(-6) M) or dexamethasone (10(-9)-10(-7) M). Gel chromatographic analysis (Sephadex G-50) revealed that the major forms of beta-END-LI released by AL cells corresponded to beta-END and beta-lipotropin (beta-LPH) in molecular size, whereas virtually all of the immunoreactivity released by NIL cells resembled beta-END. In vivo administration of dexamethasone attenuated the stress-induced release of beta-END-LI in a dose- and time-related fashion, having a more pronounced effect on plasma levels of beta-END-LI corresponding to beta-LPH in molecular size. Metyrapone (100 mg/kg), an inhibitor of glucocorticoid synthesis, evoked a rapid (20-40 min) four- to sixfold increase in total plasma beta-END-LI and 75% of this rise was due to immunoreactivity resembling beta-LPH in size. This response was diminished by coadministration of either dexamethasone (0.05-1.25 mg/kg) or corticosterone (0.05-1.25 mg/kg) and completely prevented by 4-hr pretreatment with dexamethasone (50 micrograms/kg). The briskness of the plasma beta-END-LI response to acute changes in glucocorticoid status suggests that a "rapid" feedback mechanism operates in the physiologic control of pituitary beta-END-LI secretion. Moreover, the ability of glucocorticoids to selectively inhibit AL release of beta-END-LI in vitro and their pronounced effect on plasma levels of beta-END-LI resembling beta-LPH, a marker of AL secretion, together indicate that glucocorticoids exert a selective influence over the secretion of AL corticotrophs in vivo. This demonstration of differential regulation of the AL versus IL secretion of beta-END-LI in vivo most likely reflects a phenomena having biologic importance related to the different physiologic actions of the several molecular forms of beta-END-LI secreted by the two tissues.     

Lipocorticotropic peptides in Cushing's disease: in vitro studies

The immunologic patterns of 3 human pituitary adenomas of Cushing's disease have been studied after gel exclusion chromatography (Sephadex G-50). The immunologic characteristics were examined with three radioimmunoassays specific for human corticotropin (ACTH), lipotropin (LPH) and beta-endorphin (beta-End). In cell tumor extracts, chromatographic peaks corresponding to beta-LPH, gamma-LPH, beta-End and ACTH were identified. The ACTH/beta-LP-beta-End ratio was 1 in the 3 cases. Additionally, in the 3 cases, a chromatographic peak, partially cross-reacting in the beta-End assay, was eluted after beta-End, thus suggesting the presence of a fragment of the molecule. In 1 case, a peak of large molecular weight material with N- and C-terminal beta-LPH and ACTH immunoreactivity was observed, which corresponded to the precursor material. The release and the effects of various stimuli were studied on dispersed tumor cells in primary culture. The tumor cells had a biphasic basal secretion rate with a rapid increase of ACTH/beta-LPH-beta-End in the culture medium during the first 2 h. Then the release, studied during 2 days, was slower. Chromatographic studies showed that the beta-LPH/beta-End ratio was 0.8 in the cells and 0.3 in the medium, due essentially to the release of beta-End and beta-End-like materials. The cells released ACTH and beta-LPH-beta-End in equimolar ratio after stimulation with arginine vasopressin (AVP). The maximum effect was obtained with 10(-6) M AVP (D50 = 1 10(-9) M). Dibutyryl cyclic AMP (2. 10(-3) M) induced maximal release of ACTH/beta-LPH-beta-End. This stimulation was suppressed by a 48-hour preincubation with dexamethasone (10(-8)-10(-6) M). There was no effect of TRH and LH-RH on cell release. Dopamine (10(-6) M) specifically blocked the release of ACTH/beta-LPH-beta-End in 1 case. These data showed (a) heterogeneity of chromatographic profiles from case to case; (b) the presence of material in the tumor, cell extracts and culture medium corresponding to fragment(s) of beta-End; (c) culture studies demonstrated that tumor cells remain responsive to AVP stimulation and dexamethasone suppression, and (d) the dopamine inhibition of ACTH and beta-End release needs further investigation.     

Development of the sex difference in glandular kallikrein and prolactin levels in the anterior pituitary of the rat

Glandular kallikrein is a major estrogen-induced and dopamine-repressed protein of the rat anterior pituitary that appears to originate from lactotrophs. This study examined the development of glandular kallikrein levels in the anterior pituitary in both female and male rats and compared it to anterior pituitary prolactin. In addition, the development of glandular kallikrein levels in the neurointermediate lobe of the pituitary and the kidney were also examined. During puberty, a dramatic surge in glandular kallikrein occurred in female anterior pituitaries (16- to 20-fold increase) and levels remained elevated thereafter. The dynamics of the increase were biphasic--glandular kallikrein increased between Day 30 and 45, plateaued between Days 45 and 55, and then increased again between Days 55 and 65. Female anterior pituitary prolactin increased 7- to 8-fold during puberty. The rise during puberty was biphasic and was generally synchronized with increases in glandular kallikrein. However, the initial rise was proportionately less than that of glandular kallikrein, and the secondary surge was more dramatic. In contrast to females, anterior pituitary glandular kallikrein remained at low levels in male rats; prolactin levels also remained unchanged through puberty and increased moderately thereafter. Glandular kallikrein in the female neurointermediate lobe remained unchanged through Day 55, almost doubled on Day 60, and returned to prepubertal levels by Day 65; males did not exhibit the transient surge in neurointermediate lobe levels. Starting at age 60 days, renal glandular kallikrein was found to be slightly higher (15-20%) in females than in males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypsin Activation, Partial Characterization, and Distribution of Kallikrein-Like and Thrombin-Like Proteases in the Neurointermediate Lobe of the Rat Pituitary

This study examined whether the neurointermediate lobe (NIL) of the rat pituitary contains latent kallikrein- and thrombin-like proteases activated by trypsin. Partial characterization of such proteases was attempted. Also examined were the distribution of proteolytic activity within the NIL and levels in both male and female lobes. NIL homogenates were assayed for proteolytic activity at pH 8.0 before and after incubation with trypsin (10 micrograms/ml). Trypsin caused a 10-fold activation of kallikrein-like activity and a 40-fold activation of thrombin-like activity in NIL homogenates. The kallikrein-like activity was separated into two components using diethylaminoethyl-Sephadex. The predominant kallikrein-like protease was a potent kininogenase closely related or identical to glandular kallikrein and was almost exclusively localized to the intermediate lobe. The second kallikrein-like protease (kallikrein A) was a weak kininogenase sensitive to inhibition by both soybean trypsin inhibitor and aprotinin and was similarly concentrated in both the neural lobe and the intermediate lobe. The thrombin-like protease was sensitive to inhibition by hirudin (a specific thrombin inhibitor), clotted fibrinogen, and was slightly more concentrated in the neural lobe than in the intermediate lobe. NILs from female rats contained approximately 40% less kallikrein activity than NILs from male rats but did not differ in their content of thrombin-like activity.     

Immunohistochemical localization of a thyroid hormone-binding protein (p55) in human tissues

We have localized p55, a thyroid hormone-binding protein found in the endoplasmic reticulum in cultured cells, in samples of normal human and monkey tissues, using a monoclonal antibody with cryostat sections and immunoperoxidase histochemistry. Large amounts of p55 were found in many tissues, generally corresponding to the amount of endoplasmic reticulum contained in each cell type. Intense localization of p55 was found in cells of the anterior and intermediate pituitary lobes, in epithelial cells of thyroid follicles, in the glandular epithelium of mammary gland, in hepatocytes, in Paneth cells and Brunner's glands in duodenum, in acinar cells of pancreas, in adrenal cortical cells, and in scattered interstitial fibroblastic cells in many tissues. These results suggest a potential role for thyroid hormone and p55 in regulating protein synthesis or secretion in multiple organs.